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Phase separation is a thermodynamic process, but cells are inherently out of equilibrium. Guilhas et al. (2020) identify an active process through which an ATP-dependent motor controls the number and position of biomolecular condensates in bacteria.
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Bacterias , TermodinámicaRESUMEN
Viruses compartmentalize their replication and assembly machinery to both evade detection and concentrate the viral proteins and nucleic acids necessary for genome replication and virion production. Accumulating evidence suggests that diverse RNA and DNA viruses form replication organelles and nucleocapsid assembly sites using phase separation. In general, the biogenesis of these compartments is regulated by two types of viral protein, collectively known as antiterminators and nucleocapsid proteins, respectively. Herein, we discuss how RNA viruses establish replication organelles and nucleocapsid assembly sites, and the evidence that these compartments form through phase separation. While this review focuses on RNA viruses, accumulating evidence suggests that all viruses rely on phase separation and form biomolecular condensates important for completing the infectious cycle.
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Virus ARN , Virus , Condensados Biomoleculares , Fase S , Virus/genética , Virus ARN/genética , ARNRESUMEN
Nonmembrane-bound organelles such as RNA granules behave like dynamic droplets, but the molecular details of their assembly are poorly understood. Several recent papers identify structural features that drive granule assembly, shedding light on how phase transitions functionally organize the cell and may lead to pathological protein aggregation.
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Proteínas/química , ARN/química , Ribonucleoproteínas/química , Animales , Núcleo Celular/metabolismo , Fenómenos Fisiológicos Celulares , Citoplasma/metabolismo , Humanos , Proteínas/metabolismo , ARN/metabolismoRESUMEN
Once described as mere "bags of enzymes," bacterial cells are in fact highly organized, with many macromolecules exhibiting nonuniform localization patterns. Yet the physical and biochemical mechanisms that govern this spatial heterogeneity remain largely unknown. Here, we identify liquid-liquid phase separation (LLPS) as a mechanism for organizing clusters of RNA polymerase (RNAP) in Escherichia coli Using fluorescence imaging, we show that RNAP quickly transitions from a dispersed to clustered localization pattern as cells enter log phase in nutrient-rich media. RNAP clusters are sensitive to hexanediol, a chemical that dissolves liquid-like compartments in eukaryotic cells. In addition, we find that the transcription antitermination factor NusA forms droplets in vitro and in vivo, suggesting that it may nucleate RNAP clusters. Finally, we use single-molecule tracking to characterize the dynamics of cluster components. Our results indicate that RNAP and NusA molecules move inside clusters, with mobilities faster than a DNA locus but slower than bulk diffusion through the nucleoid. We conclude that RNAP clusters are biomolecular condensates that assemble through LLPS. This work provides direct evidence for LLPS in bacteria and demonstrates that this process can serve as a mechanism for intracellular organization in prokaryotes and eukaryotes alike.
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ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/enzimología , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Imagen Individual de Molécula , Factores de Elongación Transcripcional/genética , Factores de Elongación Transcripcional/metabolismoRESUMEN
Biomolecular condensates comprise a diverse and ubiquitous class of membraneless organelles. Condensate assembly is often described by liquid-liquid phase separation. While this process explains many key features, it cannot account for the compositional or architectural complexity that condensates display in cells. Recent work has begun to dissect the rich network of intermolecular interactions that give rise to biomolecular condensates. Here, we review the latest results from theory, simulations and experiments, and discuss what they reveal about the structure-function relationship of condensates.
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Biofisica , Biopolímeros/química , Núcleo Celular/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Animales , Simulación por Computador , Citoplasma/metabolismo , Perfilación de la Expresión Génica , Humanos , Cinética , Enfermedades Neurodegenerativas/metabolismo , Polímeros/química , ARN Ribosómico/metabolismo , Ribosomas/metabolismo , Transducción de SeñalRESUMEN
In this issue of Molecular Cell, Han and Mizuuchi present evidence for a possible Turing-like reaction-diffusion mechanism underlying target immunity by the bacteriophage Mu.
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Nuclear bodies are RNA and protein-rich, membraneless organelles that play important roles in gene regulation. The largest and most well-known nuclear body is the nucleolus, an organelle whose primary function in ribosome biogenesis makes it key for cell growth and size homeostasis. The nucleolus and other nuclear bodies behave like liquid-phase droplets and appear to condense from the nucleoplasm by concentration-dependent phase separation. However, nucleoli actively consume chemical energy, and it is unclear how such nonequilibrium activity might impact classical liquid-liquid phase separation. Here, we combine in vivo and in vitro experiments with theory and simulation to characterize the assembly and disassembly dynamics of nucleoli in early Caenorhabditis elegans embryos. In addition to classical nucleoli that assemble at the transcriptionally active nucleolar organizing regions, we observe dozens of "extranucleolar droplets" (ENDs) that condense in the nucleoplasm in a transcription-independent manner. We show that growth of nucleoli and ENDs is consistent with a first-order phase transition in which late-stage coarsening dynamics are mediated by Brownian coalescence and, to a lesser degree, Ostwald ripening. By manipulating C. elegans cell size, we change nucleolar component concentration and confirm several key model predictions. Our results show that rRNA transcription and other nonequilibrium biological activity can modulate the effective thermodynamic parameters governing nucleolar and END assembly, but do not appear to fundamentally alter the passive phase separation mechanism.
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Caenorhabditis elegans , Núcleo Celular/metabolismo , ARN Ribosómico/química , Transcripción Genética , Animales , Nucléolo Celular/metabolismo , Citoplasma/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Cuerpos de Inclusión Intranucleares/metabolismo , Microscopía Fluorescente , TermodinámicaRESUMEN
Chromosomal loci jiggle in place between segregation events in prokaryotic cells and during interphase in eukaryotic nuclei. This motion seems random and is often attributed to brownian motion. However, we show here that locus dynamics in live bacteria and yeast are sensitive to metabolic activity. When ATP synthesis is inhibited, the apparent diffusion coefficient decreases, whereas the subdiffusive scaling exponent remains constant. Furthermore, the magnitude of locus motion increases more steeply with temperature in untreated cells than in ATP-depleted cells. This "superthermal" response suggests that untreated cells have an additional source of molecular agitation, beyond thermal motion, that increases sharply with temperature. Such ATP-dependent fluctuations are likely mechanical, because the heat dissipated from metabolic processes is insufficient to account for the difference in locus motion between untreated and ATP-depleted cells. Our data indicate that ATP-dependent enzymatic activity, in addition to thermal fluctuations, contributes to the molecular agitation driving random (sub)diffusive motion in the living cell.
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Adenosina Trifosfato/metabolismo , Cromosomas Bacterianos/genética , Cromosomas Fúngicos/genética , Sitios Genéticos , 2,4-Dinitrofenol/farmacología , Algoritmos , Antimetabolitos/farmacología , Cromosomas Bacterianos/metabolismo , Cromosomas Fúngicos/metabolismo , Desoxiglucosa/farmacología , Difusión , Inhibidores Enzimáticos/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Microscopía por Video , Movimiento (Física) , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Rifampin/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Azida Sódica/farmacología , Temperatura , Imagen de Lapso de Tiempo , Desacopladores/farmacologíaRESUMEN
The nucleolus is a multicomponent structure made of RNA and proteins that serves as the site of ribosome biogenesis within the nucleus. It has been extensively studied as a prototype of a biomolecular condensate whose assembly is driven by phase separation. While the steady-state size of the nucleolus is quantitatively accounted for by the thermodynamics of phase separation, we show that experimental measurements of the assembly dynamics are inconsistent with a simple model of a phase-separating system relaxing to its equilibrium state. Instead, we show that the dynamics are well described by a model in which the transcription of ribosomal RNA actively drives nucleolar assembly. We find that our model of active transcription-templated assembly quantitatively accounts for the rapid kinetics observed in early embryos at different developmental stages, and for different RNAi perturbations of embryo size. Our model predicts a scaling of the time to assembly with the volume of the nucleus to the one-third power, which is confirmed by experimental data. Our study highlights the role of active processes such as transcription in controlling the placement and timing of assembly of membraneless organelles.
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Biomolecular condensates, first discovered in eukaryotic cells, were recently found in bacteria. The small size of these organisms presents unique challenges for identifying and characterizing condensates. Here, we describe a single-molecule approach for studying biomolecular condensates in live bacterial cells. Specifically, we outline a protocol to quantify the mobility of RNA polymerase in E. coli using HILO (highly inclined and laminated optical sheet) illumination with the photoconvertible fluorophore mMaple3. Our analysis classifies the trajectories of individual molecules by their local density, enabling a comparison of molecular mobilities between different subcellular compartments.
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Escherichia coli , Imagen Individual de Molécula , ARN Polimerasas Dirigidas por ADN , Escherichia coli/genética , Células Eucariotas , ARNRESUMEN
In the past almost 15 years, we witnessed the birth of a new scientific field focused on the existence, formation, biological functions, and disease associations of membraneless bodies in cells, now referred to as biomolecular condensates. Pioneering studies from several laboratories [reviewed in1-3] supported a model wherein biomolecular condensates associated with diverse biological processes form through the process of phase separation. These and other findings that followed have revolutionized our understanding of how biomolecules are organized in space and time within cells to perform myriad biological functions, including cell fate determination, signal transduction, endocytosis, regulation of gene expression and protein translation, and regulation of RNA metabolism. Further, condensates formed through aberrant phase transitions have been associated with numerous human diseases, prominently including neurodegeneration and cancer. While in some cases, rigorous evidence supports links between formation of biomolecular condensates through phase separation and biological functions, in many others such links are less robustly supported, which has led to rightful scrutiny of the generality of the roles of phase separation in biology and disease.4-7 During a week-long workshop in March 2022 at the Telluride Science Research Center (TSRC) in Telluride, Colorado, â¼25 scientists addressed key questions surrounding the biomolecular condensates field. Herein, we present insights gained through these discussions, addressing topics including, roles of condensates in diverse biological processes and systems, and normal and disease cell states, their applications to synthetic biology, and the potential for therapeutically targeting biomolecular condensates.
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Condensados Biomoleculares , Enfermedad , Transición de Fase , HumanosRESUMEN
Single particle tracking is a powerful technique for investigating the dynamic behavior of biological molecules. However, many of the analytical tools are prone to generate results that can lead to mistaken interpretations of the underlying transport process. Here, we explore the effects of localization error and confinement on the velocity autocorrelation function, Cυ. We show that calculation of Cυ across a range of discretizations can distinguish the effects of localization error, confinement, and medium elasticity. Thus, under certain regimes, Cυ can be used as a diagnostic tool to identify the underlying mechanism of anomalous diffusion. Finally, we apply our analysis to experimental data sets of chromosomal loci and RNA-protein particles in Escherichia coli.
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Biofisica/métodos , Elasticidad , Escherichia coli/metabolismo , Modelos Biológicos , Proteínas Bacterianas/metabolismo , Transporte Biológico , Cromosomas Bacterianos/metabolismo , Difusión , Simulación de Dinámica Molecular , ARN Bacteriano/metabolismoRESUMEN
Tracking of fluorescently labeled chromosomal loci in live bacterial cells reveals a robust scaling of the mean square displacement (MSD) as τ(0.39). We propose that the observed motion arises from relaxation of the Rouse modes of the DNA polymer within the viscoelastic environment of the cytoplasm. The time-averaged and ensemble-averaged MSD of chromosomal loci exhibit ergodicity, and the velocity autocorrelation function is negative at short time lags. These observations are most consistent with fractional Langevin motion and rule out a continuous time random walk model as an explanation for anomalous motion in vivo.
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Cromosomas Bacterianos/metabolismo , Citoplasma/metabolismo , Elasticidad , Sitios Genéticos , Movimiento , Proteínas Bacterianas/metabolismo , Caulobacter/citología , Caulobacter/metabolismo , Supervivencia Celular , Difusión , Escherichia coli/citología , Escherichia coli/metabolismo , Colorantes Fluorescentes/metabolismo , Cinética , Modelos Biológicos , ARN Bacteriano/metabolismo , ViscosidadRESUMEN
The nucleolus is a prominent, membraneless compartment found within the nucleus of eukaryotic cells. It forms around ribosomal RNA (rRNA) genes, where it coordinates the transcription, processing, and packaging of rRNA to produce ribosomal subunits. Recent efforts to characterize the biophysical properties of the nucleolus have transformed our understanding of the assembly and organization of this dynamic compartment. Indeed, soluble macromolecules condense from the nucleoplasm to form nucleoli through a process called liquid-liquid phase separation. Individual nucleolar components rapidly exchange with the nucleoplasm and separate within the nucleolus itself to form distinct subcompartments. In addition to its essential role in ribosome biogenesis, the nucleolus regulates many aspects of cell physiology, including genome organization, stress responses, senescence and lifespan. Consequently, the nucleolus is implicated in several human diseases, such as Hutchinson-Gilford progeria syndrome, Diamond-Blackfan anemia, and various forms of cancer. This Special Issue highlights new insights into the physical and molecular mechanisms that control the architecture and diverse functions of the nucleolus, and how they break down in disease.
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Nucléolo Celular/fisiología , HumanosRESUMEN
Because of their unique physicochemical properties, lanthanide-doped upconverting nanoparticles (Ln-UCNPs) have exceptional potential for biological applications. However, the use in biological systems is hampered by the limited understanding of their bionano interactions. Our multidisciplinary study has generated these insights through in-depth and quantitative analyses. The Ln-UCNPs examined here are spherical, monodisperse, and stable in aqueous environments. We show that Ln-UCNPs were associated with HeLa (cervical cancer) and LLC-PK1 (renal proximal tubule) cells and were nontoxic over a wide concentration range. Multiple biomarkers were assessed to monitor the cellular homeostasis in Ln-UCNP-treated cells. To this end, we evaluated the nuclear lamina, nucleoli, and nuclear transport factors. Single-cell analyses quantified the impact on Nrf2 and NF-κB, two transcription factors that control stress and immune responses. Moreover, we measured Ln-UCNP-induced changes in the abundance of molecular chaperones. Collectively, in vitro studies confirmed that Ln-UCNPs are nontoxic and trigger minor cellular stress responses. This lack of toxicity was verified in vivo, using the model organism Caenorhabditis elegans. The compatibility with biological systems prompted us to assess Ln-UCNPs as potential contrast agents for magnetic resonance imaging. We demonstrated that the Ln-UCNPs examined here were especially suitable as T2 contrast agents; they clearly outperformed the clinically used Gadovist. Taken together, our interdisciplinary work provides robust evidence for the nontoxicity of Ln-UCNPs. This sets the stage for the translation of Ln-UCNP for use in complex biological systems.
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Enclosed by two membranes, the nucleus itself is comprised of various membraneless compartments, including nuclear bodies and chromatin domains. These compartments play an important though still poorly understood role in gene regulation. Significant progress has been made in characterizing the dynamic behavior of nuclear compartments and liquid-liquid phase separation (LLPS) has emerged as a prominent mechanism governing their assembly. However, recent work reveals that certain nuclear structures violate key predictions of LLPS, suggesting that alternative mechanisms likely contribute to nuclear organization. Here, we review the evidence for and against LLPS for several nuclear compartments and discuss experimental strategies to identify the mechanism(s) underlying their assembly. We propose that LLPS, together with multiple modes of protein-nucleic acid binding, drive spatiotemporal organization of the nucleus and facilitate functional diversity among nuclear compartments.
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Concomitant with packaging the genome, the cell nucleus must also spatially organize the nucleoplasm. This complex mixture of proteins and nucleic acids partitions into a variety of phase-separated, membraneless organelles called nuclear bodies. Significant progress has been made in understanding the relationship between the material properties of nuclear bodies and their structural and functional consequences. Furthermore, the molecular basis of these condensed phases is beginning to emerge. Here, I review the latest work in this exciting field, highlighting recent advances and new challenges.
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Nucléolo Celular/química , Nucléolo Celular/metabolismo , Núcleo Celular/química , Núcleo Celular/metabolismo , Cuerpos de Inclusión Intranucleares/química , Cuerpos de Inclusión Intranucleares/metabolismo , Animales , Humanos , Procesamiento Postranscripcional del ARN , Ribosomas/metabolismo , Transcripción GenéticaRESUMEN
Multicellular organisms must regulate their growth across the diverse length scales of biological organization, but how this growth is controlled from organelle to body, while coordinating interdependent functions at each scale, remains poorly understood. We utilized the C. elegans worm intestine as a model system to identify distinct allometric scaling laws, revealing that the growth of individual structures is differentially regulated during development. We show that the volume of the nucleolus, a subcellular organelle, is directly proportional (isometric) to cell size during larval development. In contrast to findings in a variety of other systems, the size of the nucleus grows more slowly and is hypoallometric to the cell. We further demonstrate that the relative size of the nucleolus, the site of ribosome biogenesis, is predictive of the growth rate of the entire worm. These results highlight the importance of subcellular size for organism-level function in multicellular organisms.
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Caenorhabditis elegans/crecimiento & desarrollo , Nucléolo Celular/genética , Larva/crecimiento & desarrollo , Animales , Tamaño Corporal , Caenorhabditis elegans/genética , Nucléolo Celular/metabolismo , Tamaño de la Célula , Larva/genética , Biogénesis de Organelos , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
Just as organ size typically increases with body size, the size of intracellular structures changes as cells grow and divide. Indeed, many organelles, such as the nucleus [1, 2], mitochondria [3], mitotic spindle [4, 5], and centrosome [6], exhibit size scaling, a phenomenon in which organelle size depends linearly on cell size. However, the mechanisms of organelle size scaling remain unclear. Here, we show that the size of the nucleolus, a membraneless organelle important for cell-size homeostasis [7], is coupled to cell size by an intracellular phase transition. We find that nucleolar size directly scales with cell size in early C. elegans embryos. Surprisingly, however, when embryo size is altered, we observe inverse scaling: nucleolar size increases in small cells and decreases in large cells. We demonstrate that this seemingly contradictory result arises from maternal loading of a fixed number rather than a fixed concentration of nucleolar components, which condense into nucleoli only above a threshold concentration. Our results suggest that the physics of phase transitions can dictate whether an organelle assembles, and, if so, its size, providing a mechanistic link between organelle assembly and cell size. Since the nucleolus is known to play a key role in cell growth, this biophysical readout of cell size could provide a novel feedback mechanism for growth control.